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[Preprint]. 2024 Aug 21:2024.08.19.608619.
doi: 10.1101/2024.08.19.608619.

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response

Alpha A Lee  1   2 , Isabelle Amick  1   2 , Jasmin C Aschenbrenner  1   3   4 , Haim M Barr  1   5 , Jared Benjamin  1   6 , Alexander Brandis  1   7 , Galit Cohen  5 , Randy Diaz-Tapia  1   6 , Shirly Duberstein  1   5 , Jessica Dixon  1   8 , David Cousins  1   9 , Michael Fairhead  1   8 , Daren Fearon  1   3   4 , James Frick  1   2 , James Gayvert  1   2 , Andre S Godoy  1   10 , Ed J Griffin  1   9 , Kilian Huber  1   8 , Lizbé Koekemoer  1   8 , Noa Lahav  1   5 , Peter G Marples  1   3   4 , Briana L McGovern  1   6 , Tevie Mehlman  1   7 , Matthew C Robinson  1   2 , Usha Singh  1   8 , Tamas Szommer  1   8 , Charles W E Tomlinson  1   3   4 , Thomas Vargo  1   2 , Frank von Delft  1   3   8   4 , SiYi Wang  1   8 , Kris White  1   6 , Eleanor Williams  1   8 , Max Winokan  1   3   4
Affiliations

Discovery of potent SARS-CoV-2 nsp3 macrodomain inhibitors uncovers lack of translation to cellular antiviral response

Alpha A Lee et al. bioRxiv. .

Abstract

A strategy for pandemic preparedness is the development of antivirals against a wide set of viral targets with complementary mechanisms of action. SARS-CoV-2 nsp3-mac1 is a viral macrodomain with ADP-ribosylhydrolase activity, which counteracts host immune response. Targeting the virus' immunomodulatory functionality offers a differentiated strategy to inhibit SARS-CoV-2 compared to approved therapeutics, which target viral replication directly. Here we report a fragment-based lead generation campaign guided by computational approaches. We discover tool compounds which inhibit nsp3-mac1 activity at low nanomolar concentrations, and with responsive structure-activity relationships, high selectivity, and drug-like properties. Using our inhibitors, we show that inhibition of nsp3-mac1 increases ADP-ribosylation, but surprisingly does not translate to demonstrable antiviral activity in cell culture and iPSC-derived pneumocyte models. Further, no synergistic activity is observed in combination with interferon gamma, a main protease inhibitor, nor a papain-like protease inhibitor. Our results question the extent to which targeting modulation of innate immunity-driven ADP-ribosylation can influence SARS-CoV-2 replication. Moreover, these findings suggest that nsp3-mac1 might not be a suitable target for antiviral therapeutics development.

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Figures

Figure 1:
Figure 1:. Summary of the SARS-CoV-2 nsp3-mac1 campaign.
(A) Schematic of the role of viral nsp3-mac1 in reversing ADP-ribosylation via innate immune response. (B) We report a rapid fragment-to-lead campaign, realising significant strides in potency. The inset shows the structure of a lead compound (ASAP-0008327), confirming active site engagement (PDB ID 7GZU).
Figure 2:
Figure 2:. Pharmacophore identification enables fragment-based hit discovery.
(A) Conversion of fragments into pharmacophore clusters. The red, blue and green spheres correspond to hydrogen bond acceptor, donor and hydrophobic pharmacophores, respectively. (B) Selected hit molecules superimposed on the identified pharmacophores.
Figure 3:
Figure 3:. Hit to lead chemistry via library chemistry.
We designed parallel medicinal chemistry libraries based on (A) SNAr, (B) Suzuki, and (C) Amide chemistries to realise step change in potency. (D) shows a multistep library which enables variations in the benzodioxane motif. Computational overlays between crystallographic structure of fragments (transparent) and our lead compounds (solid) provided inspiration for library design.
Figure 4:
Figure 4:. Combining structure-activity relationship furnishes potent inhibitors.
(A) shows key variations in one substituents around the scaffold (potency values are reported for the racemic mixture). (B) shows that the structure-activity relationship is synergistic; we report the IC50 of the bioactive pure enantiomer. (C) shows potency pIC50 as a function of permeability and solubility, revealing no inherent tradeoff. The dotted lines demarcate regions of high permeability (>10−6 cm/s) and solubility (>50 μM).
Figure 5:
Figure 5:. Binding interactions of lead compounds and fragments against SARS-CoV-2 nsp3-mac1.
Crystal structures of (A) ASAP-0011198 (PDB ID 7H1D), (B) ASAP-0011446 (PDB ID 7H0D) and (C) ASAP-0011184 (PDB ID 7GZZ) bound to the ADPr-binding site of nsp3-mac1 and selected fragments (D) ZINC000263392672 (PDB ID 5RSG), (E) ZINC000089254160_N3 (PDB ID 5RSJ) and (F) Z26781964 (PDB 5ST2) that were used as inspiration for the lead compounds. Dashes depict polar (black), aromatic (green), and hydrophobic (orange) interactions between ligands and nsp3-mac1. 2Fo-Fc map (σ=1.0) is displayed as blue mesh around the lead compounds. (G) Analysis of interactions by all fragment hits found in the crystallographic fragment screen against nsp3-mac1 (13).
Figure 6:
Figure 6:. Our campaign furnished a toolbox of chemical probes against SARS-CoV-2 nsp3-mac1.
The table shows key physicochemical, in vitro ADME, and selectivity data for tool compounds. These tool compounds clearly reveal that nsp3-mac1 inhibition does not translate to antiviral activity.
Figure 7:
Figure 7:. nsp3-mac1 inhibition does not translate to antiviral response.
(A) Cellular Thermal Shift Assay (CETSA) demonstrating cellular target engagement. (B) Mono-ADP-ribosylation integral mean fluorescence of infected HelaACE2 cells as a function of compound concentration, showing a statistically significant increase upon dosing ASAP-0011184. (C) No statistically significant antiviral effect is observed for ASAP-0011184 in an iPSC-derived pneumocyte system. Data was analysed by two-way ANOVA (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).
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